This invention relates to a continuous cell culture system and apparatus therefor. More particularly, this invention relates to a flask assembly which can be used for suspension culturing of mammalian cells in which fresh medium can be added and spent medium can be separated from the growing cells by filtration and withdrawn from the flask on a continuous or semi-continuous basis.
In recent years there has been rapid growth in the development of various methods for the culturing of mammalian cells in suspension. The attainment of high cell densities is a primary objective of many of these approaches. The use of a cell culture vessel with controlled agitation by means of a magnetic stirrer bar or mechanically driven impeller on a shaft is a typical feature of these methods. Examples of such apparatus are disclosed in U.S. Pat. Nos. 2,958,517; 3,039,932; 3,572,651; 3,622,129; and 3,649,465. These are essentially batch type spin culture devices in which the cells are incubated in a fixed amount of nutrient under appropriate culture conditions until cell growth has ceased.
It has been recognized that maintenance of constant levels of required nutrients coupled with removal of toxic cell by-products facilitates the propagation of cells in higher densities than is obtained in batch processes where the cells are grown in a fixed amount of nutrient and harvested after significant growth ceases. One approach to obtain such higher cell densities employs the batch type apparatus but involves daily centrifugation and resuspension of cells in fresh medium as reported by Glinos et al, J. Cell Physiol. 79, 79-90 (1971). Another approach makes use of special apparatus developed for continuous suspension cell culturing. Examples of such apparatus developed for continuous suspension cell culturing are disclosed in U.S. Pat. No. 3,647,632; by Himmelfarb, Science 164, 555-57 (1969); and by Thayer et al, "Tissue Culture Methods and Applications" (Kruse and Patterson, editors), Academic Press, pp. 345-51 (1973). The use of such devices in continuous cell culturing of various cell lines is further described by Thayer et al, Cancer Res. 30, 1709-14 (1970); Cook et al, In Vitro 9, 318-22 (1974); and by Lynn and Acton, Biotech. Bioeng. 27, 659-73 (1975).
Notwithstanding the advantages obtained with spin culture devices of the foregoing type, a problem which frequently exists is the presence of rotating bearing and seal surfaces which can contact the cells in the fluid suspension being agitated and thereby cause grinding and cell disruption at high cell densities. Placement of the rotating shaft driving means outside the culture vessel in order to avoid the grinding of cells in the flask introduces another problem, namely contamination by seepage into the vessel around the rotating shaft. Still another problem which exists with prior cell culture devices which employ filtering means is the clogging of the filter with cell debris.